The invention relates to relatively short peptides (termed I-conotoxins herein), about 30-50 residues in length, which are naturally available in minute amounts in the venom of the cone snails or analogous to the naturally available peptides, and which preferably include four disulfide bonds. The I-conotoxins are members of the I-Superfamily of conotoxins.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
Conus is a genus of predatory marine gastropods (snails) which envenomate their prey. Venomous cone snails use a highly developed projectile apparatus to deliver their cocktail of toxic conotoxins into their prey. In fish-eating species such as Conus magus the cone detects the presence of the fish using chemosensors in its siphon and when close enough extends its proboscis and fires a hollow harpoon-like tooth containing venom into the fish. This immobilizes the fish and enables the cone snail to wind it into its mouth via an attached filament. For general information on Conus and their venom see the website xe2x80x9cgrimwade.biochemxe2x80x9d at xe2x80x9cunimelb.edu.auxe2x80x9d Prey capture is accomplished through a sophisticated arsenal of peptides which target specific ion channel and receptor subtypes. Each Conus species venom appears to contain a unique set of 50-200 peptides. The composition of the venom differs greatly between species and between individual snails within each species, each optimally evolved to paralyse it""s prey. The active components of the venom are small peptides toxins, typically 10-40 amino acid residues in length and are typically highly constrained peptides due to their high density of disulphide bonds.
The venoms consist of a large number of different peptide components that when separated exhibit a range of biological activities: when injected into mice they elicit a range of physiological responses from shaking to depression. The paralytic components of the venom that have been the focus of recent investigation are the xcex1-, xcfx89- and xcexc-conotoxins. All of these conotoxins act by preventing neuronal communication, but each targets a different aspect of the process to achieve this. The xcex1-conotoxins target nicotinic ligand gated channels, the xcexc-conotoxins target the voltage-gated sodium channels and the co-conotoxins target the voltage-gated calcium channels (Olivera et al., 1985). For example a linkage has been established between xcex1-, xcex1A- and "psgr"-conotoxins and the nicotinic ligand-gated ion channel; xcfx89-conotoxins and the voltage-gated calcium channel; xcexc-conotoxins and the voltage-gated sodium channel; xcex4-conotoxins and the voltage-gated sodium channel; xcexa-conotoxins and the voltage-gated potassium channel; conantokins and the ligand-gated glutamate (NMDA) channel. For a partial list of Conus peptides and their amino acid sequences see the website xe2x80x9cpirxe2x80x9d at xe2x80x9cgeorgetown.eduxe2x80x9d.
However, the structure and function of only a small minority of these peptides have been determined to date. For peptides where function has been determined, three classes of targets have been elucidated: voltage-gated ion channels; ligand-gated ion channels, and G-protein-linked receptors.
Conus peptides which target voltage-gated ion channels include those that delay the inactivation of sodium channels, as well as blockers specific for sodium channels, calcium channels and potassium channels. Peptides that target ligand-gated ion channels include antagonists of NMDA and serotonin receptors, as well as competitive and noncompetitive nicotinic receptor antagonists. Peptides which act on G-protein receptors include neurotensin and vasopressin receptor agonists. The unprecedented pharmaceutical selectivity of conotoxins is at least in part defined by specific disulfide bond frameworks combined with hypervariable amino acids within disulfide loops (for a review see McIntosh et al., 1998).
There are drugs used in the treatment of pain, which are known in the literature and to the skilled artisan. See, for example, Merck Manual, 16th Ed. (1992). However, there is a demand for more active analgesic agents with diminished side effects and toxicity and which are non-addictive. The ideal analgesic would reduce the awareness of pain, produce analgesia over a wide range of pain types, act satisfactorily whether given orally or parenterally, produce minimal or no side effects, be free from tendency to produce tolerance and drug dependence.
Due to the high potency and exquisite selectivity of the conopeptides, several are in various stages of clinical development for treatment of human disorders. For example, two Conus peptides are being developed for the treatment of pain. The most advanced is xcfx89-conotoxin MVIIA (ziconotide), an N-type calcium channel blocker (see Heading, C., 1999; U.S. Pat. No. 5,859,186). xcfx89-Conotoxin MVIIA, isolated from Conus magus, is approximately 1000 times more potent than morphine, yet does not produce the tolerance or addictive properties of opiates. xcfx89-Conotoxin MVIIA has completed Phase III (final stages) of human clinical trials and is now awaiting U.S. Food and Drug Administration approval as a therapeutic agent. xcfx89-Conotoxin MVIIA is introduced into human patients by means of an implantable, programmable pump with a catheter threaded into the intrathecal space. Preclinical testing for use in post-surgical pain is being carried out on another Conus peptide, contulakin-G, isolated from Conus geographus (Craig et al. 1999). Contulakin-G is a 16 amino acid O-linked glycopeptide whose C-terminus resembles neurotensin. It is an agonist of neurotensin receptors, but appears significantly more potent than neurotensin in inhibiting pain in in vivo assays.
In view of a large number of biologically active substances in Conus species it is desirable to further characterize them and to identify peptides capable of treating disorders involving voltage gated ion channels, such as stroke and pain. Surprisingly, and in accordance with this invention, Applicants have discovered novel conotoxins that can be useful for the treatment of disorders involving voltage gated ion channels and could address a long felt need for a safe and effective treatment.
The invention relates to relatively short peptides (termed 1-conotoxins herein), about 30-50 residues in length, which are naturally available in minute amounts in the venom of the cone snails or analogous to the naturally available peptides, and which preferably include four disulfide bonds. The 1-conotoxins are useful for treating disorders involving voltage gated ion channels as described herein.
More specifically, the present invention is directed to 1-conotoxin peptides having the general formula I:
Xaa1-Xaa2-Xaa3-Xaa4-Cys-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Cys-Xaa11, Xaa12-Xaa13-Xaa14-Xaa15-Cys-Cys-Xaa16-Xaa17-Xaa18-Cys-Cys-Xaa19-Xaa20-Gly-Xaa21-Cys-Xaa22-Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Xaa30-Xaa30-Cys-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa40-Xaa41 (SEQ ID NO: 1), wherein Xaa1 is des-Xaa1 or Gly; Xaa2 is des-Xaa2, Pro, hydroxy-Pro (Hyp), Ala, His or Gly; Xaa3 is des-Xaa3, Ser, Val, Pro, Hyp, Thr, g-Ser (where g is glycosylation), g-Thr, g-Hyp or any synthetic hydroxylated amino acid; Xaa4 is des-Xaa4, Gly, Glu, xcex3-carboxy-Glu (Gla), Phe, Pro, Hyp, Arg, Lys, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys or any synthetic basic amino acid or Xaa4 is pyro-Glu if Xaa1, Xaa2 and Xaa3 are all des-Xaa; Xaa5 is an aliphatic amino acid bearing linear or branched saturated hydrocarbon chains such as Leu (D or L), Ile and Val or non-natural derivatives of the aliphatic amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys, any synthetic basic amino acid, Gly, Trp (D or L), neo-Trp, halo-Trp (D or L) or any synthetic aromatic amino acid; Xaa6 is Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys, any synthetic basic amino acid, Ala, an aliphatic amino acids bearing linear or branched saturated hydrocarbon chains such as Leu (D or L), Ile and Val or non-natural derivatives of the aliphatic amino acid, Thr, Ser, g-Thr or g-Ser; Xaa7 is Gly, Asp, Glu, Gla, Asn, Gln or any synthetic acidic amino acid; Xaa8 is Gly, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys, any synthetic basic amino acid, Asp, Glu, Gla, Asn, Gln or any synthetic acidic amino acid; Xaa9 is Ala, Val, Met, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys or any synthetic basic amino acid; Xaa10 is Ala, His, Ser, Thr, Pro, Hyp, g-Ser, g-Thr, g-Hyp, any synthetic hydroxylated amino acid, Asn, Gln, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys or any synthetic basic amino acid; Xaa11 is Gly, Ser, Thr, g-Ser, g-Thr, Asp, Glu, Gla, any synthetic acidic amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys or any synthetic basic amino acid; Xaa12 is Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, any synthetic aromatic amino acid, Gln, Asn or Leu (D or L); Xaa13 is Ser, Thr, g-Ser, g-Thr or His; Xaa14 is Ala, Gla, Glu, Asp, Asn, Gln, any synthetic acidic amino acid, Ser, Thr, g-Ser, g-Thr, His, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys or any synthetic basic amino acid; Xaa15 is Asp, Glu, Gla, Asn, Gln, any synthetic acidic amino acid or His; Xaa16 is des-Xaa16, Gly, His, Ser, Pro, Hyp, Thr, g-Ser, g-Thr, g-Hyp, any synthetic hydroxylated amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys or any synthetic basic amino acid; Xaa17 is des-Xaa17, His, Ser, Thr, g-Ser, g-Thr, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys, any synthetic basic amino acid, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any synthetic aromatic amino acid; Xaa18 is Val, Asn, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys or any synthetic basic amino acid; Xaa19 is des-Xaa19, Leu (D or L), Pro, Hyp, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any synthetic aromatic amino acid; Xaa20 is Gly, Ile, Ser, Thr, g-Ser, g-Thr, His, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys, any synthetic basic amino acid, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any synthetic aromatic amino acid; Xaa21 is Ser, Thr, g-Ser, g-Thr, an aliphatic amino acid bearing linear or branched saturated hydrocarbon chains such as Leu (D or L), Ile and Val or non-natural derivatives of the aliphatic amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys, any synthetic basic amino acid, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any synthetic aromatic amino acid; Xaa22 is Ala, Gln, Gla, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys or any synthetic basic amino acid; Xaa23 is Ser, Pro, Hyp, Thr, g-Ser, g-Thr, g-Hyp, any synthetic hydroxylated amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys or any synthetic basic amino acid; Xaa24 is Gln, Ser, Pro, Hyp, Thr, g-Ser, g-Thr, g-Hyp or any synthetic hydroxylated amino acid; Xaa25 is des-Xaa25, Ser, Thr, g-Ser, g-Thr or any synthetic hydroxylated amino acid; Xaa26 is des-Xaa26, Asn, Gln, Ser, Thr, g-Asn, g-Ser, g-Thr or any synthetic hydroxylated amino acid; Xaa27 is des-Xaa27, Val, Gla, Trp (D or L), neo-Trp, halo-Trp (D or L), any aromatic synthetic amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys or any synthetic basic amino acid; Xaa28 is des-Xaa28, an aliphatic amino acid bearing linear or branched saturated hydrocarbon chains such as Leu (D or L), Ile and Val or non-natural derivatives of the aliphatic amino acid; Xaa29 is des-Xaa29, an aliphatic amino acid bearing linear or branched saturated hydrocarbon chains such as Leu (D or L), Ile and Val or non-natural derivatives of the aliphatic amino acid; Xaa30 is des-Xaa30, Ile, Ser, Pro, Hyp, Thr, g-Ser, g-Thr, g-Hyp, any synthetic hydroxylated amino acid, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any synthetic aromatic amino acid; Xaa31 is des-Xaa31 or Gly; Xaa32 is Ser, Thr, g-Ser, g-Thr, Trp (D or L), neo-Trp, halo-Tip (D or L), any aromatic synthetic amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys or any synthetic basic amino acid; Xaa33 is Val, Ser, Thr, g-Ser, g-Thr, Trp (D or L), neo-Trp, halo-Trp (D or L) or any aromatic synthetic amino acid; Xaa34 is Gly, Ile, Asp, Glu, Gla, Asn, Ser, Thr, g-Asn, g-Ser or g-Thr; Xaa35 is des-Xaa35, Val, Met, Gln, Pro, Hyp, Ser, Thr, g-Ser, g-Thr, g-Hyp or any synthetic hydroxylated amino acid; Xaa36 is des-Xaa36, Val, Thr, Ser, g-Thr, g-Ser, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any synthetic aromatic amino acid; Xaa37 is des-Xaa37, Gln, Asn, Thr, Ser, g-Ser, g-Ser, g-Asn, Met, Leu, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, any synthetic aromatic amino acid, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys or any synthetic basic amino acid; Xaa38 is des-Xaa38, Leu, Ser, Thr, g-Ser, g-Thr, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys or any synthetic basic amino acid; Xaa39 is des-Xaa39, Ile, Ala, Thr, Ser, g-Ser, g-Thr, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys or any synthetic basic amino acid; Xaa40 is des-Xaa40, Asp, Lys, Arg, ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,Nxe2x80x2-dimethyl-Lys, N,Nxe2x80x2,Nxe2x80x3-trimethyl-Lys or any synthetic basic amino acid; and Xaa41 is des-Xaa41, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any synthetic aromatic amino acid, with the proviso that the peptide is not J029 as defined below. The Cys residues may be in D or L configuration and may optionally be substituted with homocysteine (D or L). The Tyr residues may be substituted with the 3-hydroxyl or 2-hydroxyl isomers and corresponding O-sulpho- and O-phospho-derivatives. The acidic amino acid residues may be substituted with any synthetic acidic amino acid, e.g., tetrazolyl derivatives of Gly and Ala. The nonnatural derivatives of the aliphatic amino acids include those synthetic derivatives bearing non-natural aliphatic branched or linear side chains CnH2n+2 up to and including n=8. The Met residues may be substituted with norleucine (Nle). The halogen is iodo, chloro, fluoro or bromo; preferably iodo for halogen substituted-Tyr and bromo for halogen-substituted Trp.
J029 has the sequence Gly-Xaa-Ser-Phe-Cys-Lys-Ala-Asp-Glu-Lys-Xaa-Cys-Glu-Tyr-His-Ala-Asp-Cys-Cys-Asn-Cys-Cys-Leu-Ser-Gly-Ile-Cys-Ala-Xaa-Ser-Thr-Asn-Trp-Ile-Leu-Xaa-Gly-Cys-Ser-Thr-Ser-Ser-Phe-Phe-Lys-Ile (SEQ ID NO:2), wherein Xaa is Pro or hydroxy-Pro.
The present invention is also directed to novel specific I-conotoxin peptides within general formula I having the mature toxin sequences set forth in Table 1. The present invention is further directed to 1-conotoxins having the amino acid sequences set forth in Tables 2-4.
In addition, the present invention is directed to the above I-contoxins in which the Arg residues may be substituted by Lys, ornithine, homoargine, nor-Lys, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any synthetic basic amino acid; the Lys residues may be substituted by Arg, ornithine, homoargine, nor-Lys, or any synthetic basic amino acid; the Tyr residues may be substituted with any synthetic hydroxy containing amino acid; the Ser residues may be substituted with Thr or any synthetic hydroxylated amino acid; the Thr residues may be substituted with Ser or any synthetic hydroxylated amino acid; the Phe and Trp residues may be substituted with any synthetic aromatic amino acid; and the Asn, Ser, Thr or Hyp residues may be glycosylated. The Cys residues may be in D or L configuration and may optionally be substituted with homocysteine (D or L). The Tyr residues may also be substituted with the 3-hydroxyl or 2-hydroxyl isomers (meta-Tyr or ortho-Tyr, respectively) and corresponding O-sulpho- and O-phospho-derivatives. The acidic amino acid residues may be substituted with any synthetic acidic amino acid, e.g., tetrazolyl derivatives of Gly and Ala. The aliphatic amino acids may be substituted by synthetic derivatives bearing non-natural aliphatic branched or linear side chains CnH2n+2 up to and including n=8. The Leu residues may be substituted with Leu (D). The Glu residues may be substituted with Gla. The Gla residues may be substituted with Glu. The N-terminal Gln residues may be substituted with pyroGlu. The Met residues may be substituted with norleucine (Nle).
The present invention is further directed to derivatives of the above peptides and peptide derivatives which are acylic permutations in which the cyclic permutants retain the native bridging pattern of native toxin. See Craik et al. (2001).
Examples of synthetic aromatic amino acid include, but are not limited to, such as nitro-Phe, 4-substituted-Phe wherein the substituent is C1-C3 alkyl, carboxyl, hyrdroxymethyl, sulphomethyl, halo, phenyl, -CHO, -CN, -SO3H and -NHAc. Examples of non-natural hydroxy containing amino acid, include, but are not limited to, such as 4-hydroxymethyl-Phe, 4--hydroxyphenyl-Gly, 2,6-dimethyl-Tyr and 5-amino-Tyr. Examples of non-natural basic amino acids include, but are not limited to, N-1-(2-pyrazolinyl)-Arg, 2-(4-piperinyl)-Gly, 2-(4-piperinyl)-Ala, 2-[3-(2S)pyrrolininyl)-Gly and 2-[3-(2S)pyrrolininyl)-Ala. These and other non-natural basic amino acids, non-natural hydroxy containing amino acids or non-natural aromatic amino acids are described in Building Block Index, Version 3.0 (1999 Catalog, pages 4-47 for hydroxy containing amino acids and aromatic amino acids and pages 66-87 for basic amino acids; see also the website xe2x80x9camino-acids.comxe2x80x9d), incorporated herein by reference, by and available from RSP Amino Acid Analogues, Inc., Worcester, Mass. Examples of non-natural acid amino acids include those derivatives bearing acidic functionality, including carboxyl, phosphate, sulfonate and non-natural tetrazolyl derivatives such as described by Ornstein et al. (1993) and in U.S. Pat. No. 5,331,001, each incorporated herein by reference.
Optionally, in the peptides of general formula I and the specific peptides described above, the Asn residues may be modified to contain an N-glycan and the Ser, Thr and Hyp residues may be modified to contain an O-glyean (e.g., g-N, g-S, g-T and g-Hyp). In accordance with the present invention, a glycan shall mean any Nxe2x80x94, Sxe2x80x94or Oxe2x80x94 linked mono-, di-, tri-, poly- or oligosaccharide that can be attached to any hydroxy, amino or thiol group of natural or modified amino acids by synthetic or enzymatic methodologies known in the art. The monosaccharides making up the glycan can include D-allose, D-altrose, D-glucose, D-mannose, D-gulose, D-idose, D-galactose, D-talose, D-galactosamine, D-glucosamine, D-N-acetyl-glucosamine (GlcNAc), D-N-acetyl-galactosamine (GalNAc), D-fucose or D-arabinose. These saccharides may be structurally modified, e.g., with one or more O-sulfate, O-phosphate, O-acetyl or acidic groups, such as sialic acid, including combinations thereof. The glycan may also include similar polyhydroxy groups, such as D-penicillamine 2,5 and halogenated derivatives thereof or polypropylene glycol derivatives. The glycosidic linkage is beta and 1-4 or 1-3, preferably 1-3. The linkage between the glycan and the amino acid may be alpha or beta, preferably alpha and is 1-.
Core O-glycans have been described by Van de Steen et al. (1998), incorporated herein by reference. Mucin type O-linked oligosaccharides are attached to Ser or Thr (or other hydroxylated residues of the present peptides) by a GalNAc residue. The monosaccharide building blocks and the linkage attached to this first GalNAc residue define the xe2x80x9ccore glycans,xe2x80x9d of which eight have been identified. The type of glycosidic linkage (orientation and connectivities) are defined for each core glycan. Suitable glycans and glycan analogs are described further in U.S. Ser. No. 09/420,797 filed Oct. 19, 1999 and in PCT Application No. PCT/US99/24380 filed Oct. 19, 1999 (PCT Published Application No. WO 00/23092), each incorporated herein by reference. A preferred glycan is Gal(xcex21xe2x86x923)GalNAc(xcex11xe2x86x92).
Optionally, in the peptides of general formula I and the specific peptides described above, pairs of Cys residues may be replaced pairwise with isoteric lactam or ester-thioether replacements, such as Ser/(Glu or Asp), Lys/(Glu or Asp), Cys/(Glu or Asp) or Cys/Ala combinations. Sequential coupling by known methods (Barnay et al., 2000; Hruby et al., 1994; Bitan et al., 1997) allows replacement of native Cys bridges with lactam bridges. Thioether analogs may be readily synthesized using halo-Ala residues commercially available from RSP Amino Acid Analogues.
The peptides of the general formula and the specific peptides disclosed herein contain 8 Cys residues leading to 4 disulfide bridges. Related peptides called Janus faced atrachatoxins (J-ACTXs) have been isolated from the Australian funnel web spider (Hadronyche versuta) (King et al., 2000). The peptides of the present invention can be aligned with these peptides as shown below for the I-Superfamily peptides R11.9 and R11.7. The alignment of all of the I-Superfamily peptides is set forth in Tables 2-4 herein.
Based on alignment of the peptides of the present invention with the J-ACTXs, the preferred disulfide bridging is as follows: Cys1-Cys6, Cys2-Cys7, Cys3-Cys-4 and Cys5-Cys8, wherein Cys1 refers to the first Cys residue in the sequence of the I-Superfamily peptides, Cys2 refers to the second Cys residue in the sequence of the I-Superfamily peptides, etc.
The present invention is also directed to the identification of the nucleic acid sequences encoding these peptides and their propeptides and the identication of nucleic acid sequences of additional related 1-conotoxin peptides. Thus, the present invention is directed to nucleic acids coding for the conotoxin peptide precursors (or conotoxin propeptides) set forth in Table 1. The present invention is further directed to the conotoxin propeptides set forth in Table 1.
The present invention is further directed to a method of treating disorders associated with voltage gated ion channel disorders in a subject comprising administering to the subject an effective amount of the pharmaceutical composition comprising a therapeutically effective amount of a 1-conotoxin peptide described herein or a pharmaceutically acceptable salt or solvate thereof. The present invention is also directed to a pharmaceutical composition comprising a therapeutically effective amount of a 1-conotoxin peptide described herein or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier.
Another embodiment of the invention contemplates a method of identifying compounds that mimic the therapeutic activity of the instant peptide, comprising the steps of: (a) conducting a biological assay on a test compound to determine the therapeutic activity; and (b) comparing the results obtained from the biological assay of the test compound to the results obtained from the biological assay of the peptide. The peptide is labeled with any conventional label, preferably a radioiodine on an available Tyr. Thus, the invention is also directed to radioiodinated 1-conotoxins.